The present invention relates to polypeptides which are useful as neovascularization inhibitors, and a method of producing them.
It is known that pathological neovascularization can be a symptom or the cause of certain diseases. An example of pathological neovascularization is the occurrence of a solid tumor. For the growth of tumor tissue beyond the diameter of 1 to 2 mm, newly formed blood vessels need to extend from the existing blood vessels to reach the tumor tissue. When the blood vessel reaches the tumor tissue, its growth is explosively accelerated (J. Folkman, J. Natl. Cancer Inst., 82:4 (1990)). On the other hand, diabetic retinopathy is accompanied with pathological neovascularization of the retina, which may lead to the loss of eyesight. Moreover, pathological neovascularization is also seen in such diseases as chronic rheumatoid arthritis, psoriasis, hemangioma, scleroderma, and neovascular glaucomas, and it is considered to be one of the main symptoms (J. Folkman and N. Engle, J. Med., 320:1211 (1989)). Therefore, it may be possible to use substances that inhibit neovascularization for the treatment of tumors and other diseases mentioned above.
Vascular endothelial cells are the cells that constitute the innermost layer of the blood vessel. Neovascularization occurs when vascular endothelial cells proliferate upon stimulation by growth factors, physiologically active substances, or mechanical damages.
Known growth factors that can directly or indirectly stimulate the proliferation of vascular endothelial cells include basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), vascular endothelial cell growth factor (VEGF), platelet-derived endothelial cell growth factor (PD-ECGF), tumor necrosis factor-xcex1 (TNF-xcex1), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor-xcex1 (TGF-xcex1), and hepatocyte growth factor (HGF) (L. Diaz-Flores et al., Histol. Histopath., 9:807 (1994)). Among these factors, vascular endothelial cell growth factor (VEGF) can be distinguished from the other growth factors by the fact that its action is very specific to vascular endothelial cells. In other words, the VEGF receptor is found in very few cells other than vascular endothelial cells.
VEGF is a glycoprotein whose molecular weight is 40,000-45,000, and exists as a dimer (P. W. Leung et al., Science, 246:1306 (1989), P. J. Keck et al., Science, 246: 1319 (1989)). VEGF acts, by binding to the VEGF receptor, to promote cell proliferation and enhance membrane permeability.
The following reports suggest the involvement of VEGF in tumor growth.
Many tumor cells secrete VEGF (S. Kondo et al., Biochem. Biophys. Res. Commun., 194:1234 (1993)). When tumor tissue sections are stained with an anti-VEGF antibody, the tumor tissue is strongly stained as well as the newly formed blood vessels surrounding it (H. F. Dvorak et al., J. Exp. Med. 174:1275 (1991), L. F. Brown et al., Cancer Res., 53:4727 (1993)). Growth of a transplanted tumor is suppressed in the mouse in which one of the VEGF receptors is genetically inactivated (B. Millauer et al., Nature, 367:576 (1994)). Anti-VEGF neutralizing antibodies exhibit anti-tumor activities in the tumor-bearing mice (K. J. Kim et al., Nature, 362:841 (1993), S. Kondo et al., Biochem. Biophys. Res. Commun., 194:1234 (1993)).
From these facts, it is considered that VEGF secreted by tumor cells play a major role in neoplastic neovascularization.
In humans there are two known VEGF receptors, FLT (M. Shibuya et al., Oncogene, 5:519 (1990), C. DeVries et al., Science, 255:989 (1992)) and KDR (B. I. Terman et al., Biochem. Biophys. Res. Commun., 187:1579 (1992)). The extracellular domain of FLT and KDR has the structure constituted by seven immunoglobulin-like domains as shown in FIG. 1. The cDNA or the soluble-type receptor of FLT has been cloned (R. L. Kendal and K. A. Thomas, Proc. Natl. Acad. Sci. U.S.A., 90:10705 (1993)). The polypeptide encoded by this cDNA corresponds to the first through to the sixth immunoglobulin-like domains of the FLT extracellular domain. This polypeptide inhibited VEGF activity by binding to VEGF with an affinity comparable to that of the full-length FLT. Regarding KDR, it is also known that the genetically engineered first through sixth immunoglobulin-like domains of the extracellular domain bind to VEGF (R. L. Kendal et al., Biochem. Biophys. Res. Commun., 201:326 (1994)).
As described above, since the mouse anti-VEGF neutralizing antibodies exhibit antitumor activity, they are expected to be useful as anti-cancer agents. However, when a mouse antibody is administered to humans, human antibodies against the mouse antibody may be produced, which could lead to neutralization of the mouse antibody or might cause anaphylactic shock. In order to avoid these undesirable effects, it is necessary to modify the amino acid sequence of the mouse antibody to be closer to that of the human antibody through chimerization (S. L. Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81:6851 (1989)) or humanization without reducing the neutralizing activity of the mouse antibody. Since this method requires advanced techniques and knowledge, experience, and labor, the results depend on individual cases and are not always successful and 100%-humanized antibodies cannot be obtained by these methods. Another method utilizes the transgenic mice that produce human antibodies for immunization (S. Wagner et al., Nucleic Acid Res., 22:1389 (1994)), but here again highly specialized techniques are required.
As described above, since the extracellular domain of the VEGF receptor specifically binds to VEGF with high affinity, thereby inhibiting the VEGF activity, it can be considered useful as an inhibitor against neovascularization. Moreover, the possibility of antibody production in a human recipient is expected to be low because it is a polypeptide of human origin. On the other hand, when a polypeptide that does not naturally exist much in the human body is administered, it is metabolized very rapidly. For example, the plasma half-life of soluble CD4, which is a receptor for HIV, is 15 minutes (D. J. Capon et al., Nature, 337:525 (1989)), and that of interferon xcex3 is 30 minutes (I. Rutenfranz and H. Kirchner, J. Interferon Res., 8:573 (1988)).
As a method for prolonging the plasma half-life, it is known to utilize a fusion polypeptide genetically engineered by combining the polypeptide of interest with a molecule having a long plasma half-life, such as an antibody molecule.
In the case of CD4, the plasma half-life was increased from 15 min to 48 hr when it was chimerized with the Fc domain of IgG1 (D. J. Capon et al., Nature, 337:525 (1989)). Such a fusion polypeptide with the Fc domain of an antibody is also expected to provide an effect to induce the effector functions that the antibody possesses, i.e., complement-dependent cytotoxicity (D. B. Amos et al., Transplantation, 7:220 (1969)) and antibody-dependent cytotoxicity (A. Y. Liu et al., Proc. Natl. Acad. Sci. U.S.A., 84:3439 (1987)). Furthermore, it is expected to drastically improve the apparent affinity when the fusion polypeptide binds to a ligand on a solid phase, such as the surface of a membrane or the extracellular matrix, since the dimerization via the Fc domain enable each molecule to bind to the ligand at two sites.
When a fusion polypeptide constructed with an antibody is utilized, it is desirable to select a polypeptide with a low molecular weight as a starting material because the molecular weight increases through the fusion. This is because, if a high molecular weight polypeptide is used, the molecular weight of the corresponding DNA is also high, which is to be handled by gene manipulation upon production of the recombinant host that produces the fusion polypeptide. In general, the larger the molecular weight of the DNA to be introduced, the less efficient the transfection of the host becomes, thereby reducing the productivity of the recombinant host. Also in general, the larger the molecular weight of the recombinant polypeptide to be produced, the smaller the amount of product tends to be. Moreover, for the treatment of solid tumors, large molecular weight polypeptides are disadvantageous as these polypeptides poorly infiltrate the diseased area (D. M. Lane et al., Br. J. Cancer, 70:521 (1994)).
The present inventors have made earnest efforts in discovering small molecular weight polypeptides among those that can inhibit neovascularization by specifically inhibiting VEGF, and particularly those which are contained in the extracellular domain of the VEGF receptor. As a result, it has been found that polypeptides containing immunoglobulin-like domain 1 and immunoglobulin-like domain 2 of the extracellular domain of KDR can inhibit the VEGF activity by specifically binding to VEGF with high affinity, thereby completing the present invention. The term xe2x80x9cpolypeptidesxe2x80x9d used herein means polypeptides constituted by amino acids that are covalently bound to each other via peptide bonds, and their lengths are not limited.
Though the polypeptide consisting of immunoglobulin-like domain 1 and immunoglobulin-like domain 2 of the extracellular domain of KDR is preferably used in the present invention because of its low molecular weight, those that contain other domains can also be used. For example, the polypeptides include the polypeptides containing immunoglobulin-like domains 1 through 3, those containing immunoglobulin-like domains 1 through 4, and those containing immunoglobulin-like domains 1 through 5. The polypeptides of the invention also include those containing immunoglobulin-like domains 1 through 5 that are deficient in any one or two domains within immunoglobulin domains 3 through 5. The amino acid sequence of the polypeptides of the present invention may be modified partially by substitution or the like as long as the polypeptides can inhibit the VEGF activity by binding to VEGF. One skilled in the art could readily perform such modification of the amino acids by known methods. As a matter of course, the polypeptides such as those containing immunoglobulin-like domains 1 through 6 and those containing immunoglobulin-like domains 1 through 7 can bind to VEGF specifically with high affinity. However, their molecular weights are too high to achieve the object of the present invention that xe2x80x9cthe polypeptide is easily expressed by recombinant DNA techniques and rapidly infiltrates into the diseased area.xe2x80x9d Although the borders between adjacent domains of KDR are not clearly determined, each domain is defined herein as the one that contains the amino acid sequence designated by the following amino acid residue numbers within the entire amino acid sequence of KDR represented by SEQ ID NO: 14, which has been already published. The amino acid residue numbers are the same as those shown in SEQ ID NO: 14. Namely, they correspond to the residue numbers counted from the amino-terminal xe2x80x9cAlaxe2x80x9d of the mature KDR, which is position 1 in the SEQ ID NO: 14.
Immunoglobulin-like domain 1: 1-115
immunoglobulin-like domain 2: 116-214
Immunoglobulin-like domain 3: 218-319
Immunoglobulin-like domain 4: 319-392
Immunoglobulin-like domain 5: 393-533
Immunoglobulin-like domain 6: 534-645
Immunoglobulin-like domain 7: 646-750
Furthermore, the present invention includes the polypeptides constructed by fusing the above extracellular domain of KDR with another protein (such as the Fc domain of immunoglobulins).
These peptides can be produced by the following procedures. A total RNA is extracted by the acid phenol method (P. Chomzynski and N. Sacchi, Anal. Biochem., 162:156 (1987)) from the cultured human vascular endothelial cells, such as human umbilical chord-derived vascular endothelial cells (commercially available from Iwaki Glass, Morinaga Dairy Products, or Kurabo), and purified into a poly A+ RNA using an oligo dT cellulose. A single-stranded or double-stranded cDNA is synthesized using this RNA as a template, reverse transcriptase, and the oligo dT (12-16) primer. The poly A+ RNA and the cDNA can be prepared in accordance with J. Sambrook et al., xe2x80x9cMolecular Cloningxe2x80x9d (Cold Spring Harbor Laboratory Press, 1989). Alternatively, the commercially available poly A+ RNA preparation reagents (oligotex-dT30, Takara) or cDNA synthesis kit (Pharmacia Biosystem) can be used. If a KDR CDNA has been already cloned from a cDNA library, the DNA corresponding to the region to be expressed can be isolated by digestion with appropriate restriction enzymes and introduced directly into an expression vector.
Next, a desired part of the DNA can be amplified by PCR using the cDNA obtained above as the template (Michael A. Innis et al., xe2x80x9cPCR protocolsxe2x80x9d, Academic Press Inc., 1990). For instance, the following primers may be used. The primer DNA can be synthesized with a DNA synthesizer (Applied Biosystems, Japan Millipore Ltd., etc.) or custom-made (Sawadee Technology). For example, in the case of obtaining a cDNA encoding immunoglobulin-like domains 1 through 6, the following primers can be used:
upstream primer:
5xe2x80x2 N(3-5) X(6) ATGGAGAGCAAGGTGCTGCTG (SEQ ID NO: 2)
downstream primer:
5xe2x80x2 N(3-5) Y(6) ACGCTCTAGGACTGTGAGCTG (SEQ ID NO: 3).
In the case of obtaining a CDNA encoding immunoglobulin-like domains 1 through 3, the following primers can be used:
upstream primer:
5xe2x80x2 N(3-5) X(6) ATGGAGAGCAAGGTGCTGCTG (SEQ ID NO: 2)
downstream primer:
5xe2x80x2 N(3-5) Y(6) AGATTCCATGCCACTTCCAAA (SEQ ID NO: 4).
In the above sequences, N stands for A, C, G, or T; X or Y stands for a restriction enzyme recognition sequence; and the numeral in the parentheses indicates the number of nucleotides. Specifically, N(3-5) means that there are 3 to 5 nucleotides of A, C, G, and T, and X(6) or Y(6) indicates the recognition site for a 6-base cutter restriction enzyme. It is desirable to choose sequences that are found in neither the DNA fragment to be amplified nor the vector to which the fragment will be inserted as the restriction enzyme recognition sequences in the above. Referring to the nucleotide sequence shown in SEQ ID NO: 1, the downstream primers can be appropriately designed to amplify the DNA fragments encoding the desired carboxy-termini. When inserted into an expression vector, it should be noted that the polypeptide-coding sequences must be placed under the control of the promoter sequence. Parts of the primer sequences which correspond to the flt DNA sequence do not need to be exactly limited to 21 bases, but could be about 17-25 bases. Although the condition for PCR can be a standard one as described in the xe2x80x9cPCR Protocolsxe2x80x9d above, the reaction may be optimized to achieve a better efficiency by appropriately changing various parameters (e.g., Mg2+ concentration, annealing temperature, extension time, the number of cycles, etc.), since the reaction proceeds differently depending on the template quantity and the primer sequences. As the DNA polymerase used for PCR, Pfu polymerase (Stratagene), which possesses a proofreading (3xe2x80x2 exonuclease) activity, or Taq polymerase supplemented with Pfu polymerase will provide a better fidelity during the PCR amplification than Taq polymerase alone (W. M. Barnes, Proc. Natl. Acad. Sci. U.S.A., 91:2216 (1994)).
Because the sequence of the DNA fragment to be amplified by PCR is known in this case, whether the desired DNA fragment has been obtained can be determined by, after amplification, confirming its size by agarose gel electrophoresis, recovering the fragment from the gel, digesting it with appropriate restriction enzymes, and examining the resulting electrophoresis pattern. Agarose gel electrophoresis, recovering of DNA fragments from the gel, and restriction enzyme digestions can be done according to the xe2x80x9cMolecular Cloningxe2x80x9d above. A commercial kit which utilizes glass beads (for example, BIORAD Prep-A-Gene) can be used for recovering DNA from a gel.
The recovered DNA fragment is digested with the restriction enzymes capable of cutting X(6) and Y(6) on both ends, deproteinated by the phenol treatment, ethanol-precipitated, and resuspended in an appropriate buffer, such as TE (10 mM Tris-HCl (pH 7.5)/1 mM EDTA). Similarly, the cloning sites of an appropriate expression vector are digested with the restriction enzymes capable of cleaving X(6) and Y(6), agarose gel electrophoresis is performed, and the vector DNA is recovered. Through this procedure, a small fragment between the X(6) and Y(6) recognition sites is eliminated. The DNA fragment to be inserted and the digested vector DNA are mixed at a ratio of, for example, vector DNA:DNA insert=1:5 to 1:10, and ligated using T4 DNA ligase. The ligation product is then added to competent E. coli cells, the transformation of the cells is performed, and transformants are screened by the antibiotic resistance on a culture medium containing the antibiotic corresponding to the selection marker (e.g., ampicillin resistance, kanamycin resistance, etc.) encoded by the vector.
The recombinant expression vector, to which the DNA fragment has been inserted, can be selected by examining the restriction enzyme digestion patterns of the plasmids in the antibiotic-resistant transformants. Alternatively, whether a transformant is a recombinant or not can be examined by performing a PCR reaction on the whole bacteria as the template, using the same set of primers as used to amplify the insert DNA, and detecting the presence or absence of the amplified target fragment. These series of procedures to obtain recombinant E. coli can be performed according to the xe2x80x9cMolecular Cloningxe2x80x9d above.
A variety of hosts can be used in order to produce the polypeptides of the present invention. For example, Gram negative and Gram positive bacteria such as Escherichia coli, bacteria belonging to the genus Pseudomonas, Bacillus subtilis, Bacillus brevis, Bacillus liqueniformis, and Bacillus thuringenesis; yeast such as Pichia pastoris, Schizosaccharomyces pombe, and Saccharomyces cerevisiae; Eumycetes such as belonging to the genus Aspergillus; insect cells such as Sf9 (derived from Spodoptera frugiperda), Sf21, TN5 (derived from Trichoplusia ni), and BN4 (derived from Bombyx mori); and mammalian cells such as CHO (derived from the Chinese hamster ovary) and COS (derived from the monkey kidney). The vector can be selected based on the suitability to the host cells. The final transformants may be easily obtained by producing the recombinant DNA first in E. coli using a shuttle vector functioning in the host to be used for production of the polypeptide of the present invention and E. coli. The transformations method used for obtaining the recombinant host that produces the polypeptide of the present invention include the competent cell method for E. coli; the competent cell method (K. Bott and G. A. Wilson, J. Bacteriol., 94:562 (1967)) and the protoplast method (M. Mandel and A. Higa, J. Mol. Biol., 53:159 (1970)) for bacteria belonging to the genus Bacillus; the protoplast method (M. Broker et al., BioTechniques, 5:516 (1987)) for yeast; and the lipofectin method (R. W. Malone et al., Proc. Natl. Acad. Sci. U. S. A., 86:6077 (1989)) and the calcium phosphate method (F. L. Graham and A. J. van der Eb, virology, 52:456 (1973)) for insect cells and mammalian cells. In addition, the electroporation method (refer to the BIORAD Company""s brochure) can be used with all the cell types described above.
Basically, the DNA encoding the region to be expressed can be inserted into the plasmid or viral DNA capable of replicating in the host downstream from a strong promoter that functions in the host. If the gene to be expressed is missing the translation initiation codon, it needs to be added. When a prokaryotic cell is used as the host, the ribosome binding sequence (J. R. MacLaughlin et al., T. Biol. Chem., 256:11283 (1981)) is necessary. It is also possible to apply a method using a vector, which is not replicapable in the host and contains a part of the host chromosomal DNA, to effect a homologous recombination with the host chromosome, thereby integrating the vector into the host chromosome (JP-A-Hei 4-278092, D. J. King et al., Biochem. J., 281:317 (1992)). On the other hand, animal or plant bodies may be used as hosts instead of cultured cells. For example, compared with the case that cultured cells are used as hosts, the polypeptide may be recovered from the body fluid of the silkworms more efficiently by constructing a recombinant virus from BmNPV, which is a silkworm virus, and inoculating it into silkworms (H. Kawai and Y. Shimomura, Bioindustry, 8:39 (1991)). The recombinant polypeptide may be obtained by transplanting the mouse myeloma cells transformed with a recombinant pSV vector into the abdominal cavity of a SCID or nude mouse and recovering the polypeptide from the abdominal fluid of the mouse. It may also be possible to use as hosts transgenic animals (G. Wright et al., Bio/Technology, 9:830 (1991)) or transgenic plants (M. Owen et al., Bio/Technology, 10:790 (1992)) constructed with the DNA of the present invention.
In order to secrete the polypeptide of the present invention extracellularly, the signal peptide coding region of KDR can be used as it is if a eukaryotic cell is used as the host. If a bacterium is used as the host, the DNA encoding the signal peptide of a host""s secreted polypeptide may be utilized. For example, the DNA encoding the signal peptide includes, as those for used in E. coli, outer membrane proteins such as OmpA or OmpF, phosphatases such as PhoA, and maltose binding protein MalB; as those for use in the genus Bacillus, the DNA encoding the signal peptide for amylases, alkaline phosphatases, and serine proteases, whose nucleotide sequences are known. If intracellular expression is desired, the signal peptide coding region except the initiation codon can be excluded. When an exogenous polypeptide is expressed at a high level in bacterial cells, inclusion bodies are often formed. If this is the case, the inclusion bodies are dissolved in an 8 M urea solution, diluted to a polypeptide concentration of several xcexcg/ml, and then dialyzed to gradually remove the urea, thereby recovering several percents of activity of the polypeptide. It is also possible to suppress the formation of inclusion bodies by concurrently expressing E. coli thioredoxin at a high level in the bacterial cells.
The polypeptide of the present invention produced by the methods as described above can be purified through usual biochemical means, including, for example, ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and hydrophobic chromatography. Since the polypeptide of the present invention has affinity to heparin, the affinity chromatography with heparin resin can be utilized. When it is produced as a fusion polypeptide with another polypeptide, it can be purified by taking advantage of the properties possessed by partner polypeptide (M. Uhlen et al., Methods Enzymol., 185:129 (1990)). For example, the purification can be carried out by affinity chromatography (F. H. Arnold, Bio/Technology, 9:151 (1991)) with protein A-Sepharose or protein G-Sepharose if the partner for the fusion polypeptide is the Fc domain of an antibody (E. Harlow and D. Lane, xe2x80x9cAntibodiesxe2x80x9d, Cold Spring Harbor Laboratory Press, 1988), with glutathione-Sepharose if it is glutathione transferase (GST) (D. B. Smith and F. S. Johnson, Gene, 67:31 (1988)), with chloramphenicol-Sepharose if it is chloramphenicol, and with Ni2+-NTA (nitryltriacetic acid)-agarose if it is a histidine oligomer.
The fractions containing the polypeptide of the present invention can be detected by EIA or western analysis using antibodies reactive with the polypeptide. The antibodies reactive with the polypeptide of the present invention can be obtained by synthesizing the oligopeptide corresponding to the N-terminal 25-39 amino acid residues, conjugating with carrier proteins such as bovine serum albumin and KLH (keyhole lymphet hemocyanin), and immunizing rabbits or other animals using a standard method (E. Harlow and D. Lane, xe2x80x9cAntibodiesxe2x80x9d, Cold Spring Harbor Laboratory Press, 1988). It is also possible to obtain the antibodies reactive with the polypeptide of the present invention by producing in E. coli a fusion protein between the polypeptide of the present invention and another polypeptide, purifying the fusion protein by taking advantage of the partner polypeptide""s properties, and by using it as the immunogen.
Since the polypeptide of the present invention binds to VEGF, this activity can be used as an index for the purification process. For example, a solution containing the non-purified polypeptide of the present invention is appropriately diluted and a 96-well polystyrene microtiter plate is coated with the solution, followed by blocking in the same manner as in preparing a antibody-coated plate for EIA. Since the thus-obtained plate specifically binds to VEGF, the binding can be detected by measuring the residual radioactivity in the wells using the 125I-labeled VEGF. A fraction from the chromatography used for purifying the polypeptide of the present invention is preincubated with 125I-VEGF and the mixture is placed into the wells of the plate to measure the residual radioactivity. If the fraction contains the polypeptide of the present invention, its presence can be confirmed because it will bind to VEGF during the preincubation, which will cause a competition with the polypeptide of the present invention on the surface of the plate, thereby reducing the biding of VEGF to the plate.
The polypeptide of the present invention inhibits the binding of VEGF to the VEGF receptor by binding to VEGF. Since the polypeptide of the present invention inhibits the VEGF activity, it blocks the proliferation of vascular endothelial cells caused by the VEGF stimulation and the enhancement of vascular permeability caused by VEGF. Furthermore, the polypeptide of the present invention blocks the neovascularization in vivo caused by VEGF, thereby inhibiting the tumor growth.
Therefore, the polypeptide of the present invention is useful as an agent for diagnosis or test of cancer and other diseases as well as a therapeutic agent for these diseases.